Influenza is an acute febrile disease caused by respiratory infection with influenza virus. Influenza viruses are classified into types A, B and C based on their core proteins. Hosts, epidemiology and clinical features somewhat differ between influenza types A, B and C. Influenza viruses are spherical viruses having a diameter of 80-120 nm, and are divided into various subtypes based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins on the surface of the virus. In the case of type A influenza, 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1 to N9) have been identified. Thus, theoretically, 144 subtypes of influenza A virus (e.g., H1N1, H1N2, etc.) exist (Treanor J. J. et al., In Principles and Practice of Infectious Diseases, 2060-85, 2005).
Influenza vaccines include inactivated vaccines and live vaccines. Inactivated vaccines are produced by purifying viruses cultured in embryonated eggs and inactivating the cultured viruses with formalin or the like. Inactivated vaccines include inactivated whole-virus vaccines, split vaccines produced by disrupting viral envelopes with ether or the like, subunit vaccines obtained by purifying the hemagglutinin and neuraminidase components, etc. As live vaccines, live attenuated influenza vaccines (LAIVs) have been developed and used. Because whole virus vaccines cause side effects in infants, these vaccines are hardly used in many countries, including Korea, and are used only in some countries. However, component vaccines such as split vaccines or subunit vaccines are highly safe and have recognized effects, and thus have been most frequently used. In addition, vaccines containing an immune adjuvant (such as MF-59) for enhancing immune responses, or virosome vaccines that form virus-like vesicles, have been developed and used in some countries (Bridges C. B. et. al., In Vaccines, 259-290, 2008; Belshe R. B. et. al., In Vaccines, 291-309, 2008).
Until now, for the production of viruses for producing vaccines against influenza, a method of inoculating seed virus into fertilized eggs and culturing the inoculated virus has been used (Korean Patent Laid-Open Publication No. 10-2012-0103737A). However, this method has very low efficiency due to problems, including the security of supply of fertilized eggs, allergic induction, and viral propagation. To obtain specific pathogen-free eggs that are used for vaccine production, chickens should be raised in germ-free facilities completely isolated from the outside in a state in which an antibiotic and a vaccine are not administered to the chickens, and fertilized eggs should be produced from the chickens. The produced pathogen-free eggs are hatched in a hatchery for about 10 days, after which virus is inoculated into the embryo or allantoic fluid of the eggs by a syringe needle or the like. The inoculated virus is cultured for 3 days after inoculation, and then the cultured virus is recovered and subjected to a purification process. The virus prepared by such procedures is subjected to an inactivation process in some cases, and then an adjuvant for inducing an effective immune response, a stabilizer, a preservative and the like are added thereto, thereby producing a vaccine. The produced vaccine is filled into individual vials or syringes, after which it is inspected, packaged and shipped. In the case of Japanese encephalitis virus, the virus is also inoculated into the brain of suckling mice in addition to fertilized eggs and cultured.
In such conventional vaccine production methods, there is difficulty in expanding production facilities. For example, additional supply of fertilized eggs as a raw material is required to expand production processes that use fertilized eggs, and for this supply, chicken farming facilities free of germs should be expanded. Because chickens raised in these facilities are immunologically very weak, these chickens are difficult to raise, compared to general chickens, and need to be thoroughly controlled. Thus, there are many limits to the expansion of such facilities, in spatial or economic terms. In addition, there may be difficulty in steadily supplying pathogen-free eggs when avian infectious diseases such as avian influenza (AI) or Newcastle disease (ND) are prevalent.
In an attempt to overcome such conventional problems, methods of producing vaccines by animal cell culture have received attention long ago (Korean Patent Laid-Open Publication No. 10-2012-0033334). These methods include a method of producing a vaccine by culturing a large amount of animal cells under germ-free conditions and infecting the cultured animal cells with virus, a method of producing only antigens, which induce antibody production, by a genetic engineering method, etc. The biggest advantage of the method of producing vaccines by animal cell culture is that the production scale can be expanded. Specifically, the production scale can be expanded as desired according to the culture scale of animal cells that are used as a raw material for vaccine production.
However, despite such many advantages, the production of vaccines by animal cell culture is not easy to achieve. This is because the initial investment is too high, a personal infrastructure for smoothly performing this production is insufficient, and the yield per unit volume is somewhat lower than that of the use of fertilized eggs or animals. In order to overcome low yields per unit volume when producing vaccines using animal cells, there were some attempts to develop excellent host animal cell lines using genetic engineering techniques (fang J. et al., Appl. Microbiol. Biotechnol, 85:1509-1520, 2010), but such attempts still remain at an insufficient level. Thus, to optimize virus production, the identification of a virus-producing cell line, the improvement of culture conditions and the improvement of infection conditions are required.
Examples of typical cell lines that are currently used for the production of viral vaccines include MDCK (cells derived from the Madin-Darby canine kidney), PerC6 (cells derived from human embryonic retinal cells genetically modified by inserting the E1 genes from the human adenovirus type 5) developed by CRUCELL (Netherland), VERO (cells derived from epithelial cells of kidney from African green monkey (Cercopithecus aethiops) isolate at the Chiba University in Chiba, Japan), and BHK21 (Cells immortalized from baby hamster kidney cells).
Meanwhile, when virus infects the body, the proliferation of the virus in the infected cells occurs, and the proliferated virus particles bud out through the cell membrane to infect other surrounding cells. In part of the defense mechanism of cells against viral infection, Bst-2 gene is expressed in the cell membrane. Bst-2 has cell membrane-binding sites at both the N-terminus and the C-terminus, and thus is expressed in the cell membrane in a form in which the middle is lifted, like a bridge. The C-terminus of Bst-2 is located in the lipid raft region of the cell membrane, and the N-terminus is located in the non-lipid raft region.
The function of the C-terminal region fixed to the lipid raft region is not yet known. Meanwhile, when virus buds from the lipid raft region to the outside of the cells, Bst-2 inhibits the passage of virus particles through the cell membrane by its region fixed to the non-lipid raft region. For this defense mechanism of mammal cells, some viruses produce a protein that promotes the degradation of the Bst-2 gene in order to avoid this mechanism of the host cells. This paradoxically indicates that the function of the Bst-2 gene strongly contributes to the inhibition of production of virus. However, not all viruses have this function, and some viruses (particularly HIV virus) have a function of promoting the degradation of the Bst-2 gene by the Vpu gene, and other most viruses do not have the avoidance mechanism that targets the Bst-2 gene.
Thus, in order to efficiently produce a large amount of virus and use the produced virus for vaccine production, there is a need for the development of a technology capable of inactivating or inhibiting the function of the Bst-2 gene in animal cells for culturing a virus that does not promote the degradation of the Bst-2 gene.
Accordingly, the present inventors have made extensive efforts to develop a method for increasing the ability of a host cell to produce a virus, and as a result, have found that, when the function of the Bst-2 gene in a cell is lacked, a virus-producing cell line that has an increased ability to produce virus, due to the promotion of extracellular release of virus and the reduction of apoptosis, can be produced, thereby completing the present invention.
The information disclosed in the Background Art section is only for the enhancement of understanding of the background of the present invention, and therefore may not contain information that forms a prior art that would already be known to a person of ordinary skill in the art.